Rabies causes an estimated 55,000 human deaths globally each year, 23,750 of which occur in Africa (Knobel et al., 2005, Bull World Health Organ 83:360-368). Moreover, 11 million people undergo rabies postexposure prophylaxis (PEP) worldwide each year. Rabies is a zoonotic disease with dogs remaining the principal host in Asia, parts of America, and large parts of Africa, and rabid dogs are the cause of most human rabies cases (Hampson et al., 2009, PLoS Biol 7:e53). It is believed that between 30% to 60% of the victims of dog bites are children under the age of 15. Inappropriate dog vaccination programs, limited access to vaccination, and postexposure treatment of individuals that have been exposed to rabid dogs are major problems in developing countries.
Rabies virus (RV), a negative-stranded RNA virus of the rhabdoviridae family, has a relatively simple, modular genome that encodes 5 structural proteins: a RNA-dependent RNA polymerase (L), a nucleoprotein (N), a phosphorylated protein (P), a matrix protein (M), and an external surface glycoprotein (G). The N, P, and L together with the genomic RNA form the ribonucleoprotein complex (RNP). The main feature of rabies virus is neuroinvasiveness, which refers to its unique ability to invade the central nervous system (CNS) from peripheral sites. Virus uptake, axonal transport, transsynaptic spread, and the rate of viral replication are key factors that determine the neuroinvasiveness of a RV (Dietzschold et al., 1983, Proc Natl Acad Sci USA 80:70-74; Dietzschold et al., 1987, Proc Natl Acad Sci USA 84:9165-9169; Morimoto et al., 2000, J Neurovirol 6:373-381; Morimoto, et al., 1999, J Virol 73:510-518). The regulation of viral replication also appears to be one of the important mechanisms contributing to RV pathogenesis. Pathogenic RV strains replicate at a lower rate than attenuated strains, which helps preserve the structure of neurons that is used by the viruses to reach the CNS. In addition, the lower expression levels of viral antigens, in particular the RV G, which is the major viral antigen responsible for the induction of protective immunity, hinders early detection by the host immune system (Morimoto, et al., 1999, J Virol 73:510-518). In contrast to wildlife RVs, most attenuated RV strains replicate very quickly and express large amounts of G, thereby inducing strong adaptive immune responses that result in virus clearance. These properties provide the basis for the use of attenuated RV strains for the pre- and PEP of rabies. A live-attenuated RV vaccine is likely to provide effective immunization with a single dose, which has practical, cost, and logistical advantages over conventional multi-dose vaccines with respect to the worldwide eradication of dog rabies. In addition, because live-attenuated RV vaccines are capable of inducing immune responses that can clear virulent RVs from the CNS (Phares et al., 2006, J Immunol 176:7666-7675; Roy et al., 2008, J Neurovirol 14:401-411), there is the possibility that such vaccines could serve as the foundation for the treatment of early stage human rabies.
Apart from efficacy, the most important prerequisite for the use of live-attenuated RV vaccines in both preexposure and postexposure immunization against rabies is safety. In this respect, the availability of reverse genetics technology, which allows the modification of viral elements that account for pathogenicity and immunogenicity, has made the systematic development of safer and more potent modified-live rabies vaccine feasible. For example, the pathogenicity of fixed RV strains (i.e., ERA, SAD) can be completely abolished for immunocompetent mice by introducing single amino acid exchanges in their G (Faber et al., 2005, J Virol 79:14141-14148), and RVs containing a SADB19 G with an Arg333→Glu333 mutation are nonpathogenic for adult mice after intracranial/intracerebral (i.c.) inoculation, and that an Asn194→Ser194 mutation in the same gene prevents the reversion to pathogenic phenotype (Faber et al., 2005, J Virol 79:14141-14148; Dietzschold et al., 2004, Vaccine 23:518-524). The G containing both mutations has been designated as GAS. Using the GAS gene, the single and double GAS RV variants, SPBNGAS and SPBNGAS-GAS, respectively, were constructed (Faber et al., 2005, J Virol 79:14141-14148; Li et al., 2008, Vaccine 26:419-426). The introduction of a second G gene significantly improves the efficacy of the vaccine by enhancing its immunogenicity through higher expression of G (Faber et al., 2002, J Virol 76:3374-3381). Elevated G expression is associated with the strong up-regulation of genes related to the NFκB signaling pathway, including IFN-α/β and IFN-γ (Li et al., 2008, Vaccine 26:419-426) and increased cell death (Faber et al., 2002, J Virol 76:3374-3381). Furthermore, the presence of two G genes also decreases substantially the probability of reversion to pathogenicity because the nonpathogenic phenotype determined by GAS is dominant over a pathogenic G that could emerge during virus growth in vivo or in vitro (Faber et al., 2007, J Virol 81:7041-7047).
Controlling rabies virus infection in domestic and wildlife animals, therefore, not only reduces the mortality in these animals but also reduces the risks of human exposure. Pre-exposure vaccinations for people who are constantly at risk further prevent human rabies, as do post-exposure immunizations for people who are bitten by rabid or suspected rabid animals. A recombinant vaccinia virus expressing rabies virus glycoprotein (VRG) has been used to control rabies in wildlife. Inactivated rabies virus vaccines are used to immunize domestic animals, particularly pets. Purified and inactivated rabies virus vaccines are used for humans in the pre- or post-exposure settings. Although these vaccines are effective, annual vaccinations are required to maintain adequate immunity in pets. For humans, multiple doses of the inactivated tissue culture vaccines are required to stimulate optimal immune responses. Furthermore, current tissue culture vaccines are expensive; thus most people in need of vaccinations (in developing countries) cannot afford them. Hence, there is a need to develop more efficacious and affordable rabies virus vaccines.